Inorganic arsenic in water and food are global health problems. Increasing epidemiologic and experimental evidence supports the role of low-moderate inorganic arsenic exposure as a cardiovascular disease (CVD) risk factor. In the Strong Heart Study (SHS), baseline urine arsenic concentrations were associated with incident CVD, supporting the need to investigate relevant mechanisms for arsenic related CVD, including epigenetic modifications. Objective: To investigate (1) if DNA epigenetic modifications mediate the association between arsenic and CVD and (2) if genetic variability modifies epigenetic mediation of arsenic related CVD in 3,574 SHS participants 45-74 years old and free of CVD at baseline. Preliminary studies: In a pilot study in the SHS, arsenic metabolism, measured by the relative proportion of arsenic species in urine, was associated with global DNA methylation and hydroxymethylation and arsenic exposure was associated with a hypomethylated region of AS3MT, the gene that codes a major methyltransferase involved in arsenic metabolism. In linkage and fine-mapping studies, genetic variants in the AS3MT region of the genome were associated with urine measures of arsenic metabolism. Design and setting: Population-based prospective cohort study of American Indian men and women from Arizona, Oklahoma and North/South Dakota recruited in 1989-1991 and followed through 2008 as part of the SHS. Data collection: Urine arsenic measures (reflecting long-term exposure), DNA samples to measure epigenetic modifications and genetic polymorphisms, CVD follow-up including coronary heart disease, stroke, peripheral artery disease and carotid plaque, and extensive data characterizing CVD and its risk factors are available. Epigenetic assessment: We will measure genome- wide blood DNA methylation at baseline using state-of-the-art high throughput technology to identify specific DNA methylation that may mediate the relationship between arsenic and incident CVD endpoints and validate the most promising regions using bisulfite pyrosequencing. Genetic assessment: We will measure 96 SNPs previously related to arsenic metabolism and toxicity in the Strong Heart Family Study, conducted in the same communities as the SHS. SNPs in candidate genes related to CVD are already available in the SHS. Statistical analysis: To evaluate if DNA epigenetic modifications mediate the association between arsenic and CVD, the following conditions will need to be met: (1) arsenic is associated with CVD (already confirmed), (2) arsenic is associated with DNA methylation, (3) DNA methylation is associated with CVD, conditional on arsenic exposure, and (4) attenuation of the arsenic-CVD association conditional on DNA methylation. Gene- epigene interactions will be assessed via general linear models and likelihood ratio tests. Significance: By investigating the contribution of arsenic epigenetics to CVD, this study can reveal novel mechanisms for arsenic health effects, identify susceptible populations, and inform risk assessment, with implications for the prevention and control of arsenic exposure in drinking water and food in the US and abroad.